Plasminogen activators (PAs) are serine proteases with trypsin-like specificity that convert the proenzyme plasminogen to the enzyme plasmin. Plasmin, in turn, is the primary agent of fibrinolysis in the blood stream where it degrades the fibrin network of a blood clot to form soluble products. The two known primary human plasminogen activators are tissue plasminogen activator (t-PA) and single chain urokinase plasminogen activator (SCU-PA), the latter being the proenzyme of urokinase (UK).
T-PA, a 63-65K protein, is secreted by numerous mammalian endothelial cells including aortic and venous endothelial cells. T-PA also has a high affinity for fibrin. For these reasons, t-PA is thought to be generated and to act directly at the site of a thrombus. SCU-PA, a 55K single chain protein, on the other hand, is thought to arrive at the site of a thrombus via the blood stream.
T-PA and SCU-PA, UK and streptokinase (SK, a third plasminogen activator), as well as modifications thereof, are under intense study to determine their respective physiological roles in thrombolysis, angiogenesis, metastasis, inflammation and ovulation. Such studies include clinical studies of the use of these substances in treating thrombosis. Such investigations also include the search for means for producing larger quantitites of these enzymes from normal human cells for use as thrombolytic agents. Additionally, such increased levels of production of the enzymes themselves would also give rise to elevated levels of the respective messenger RNA's (m RNA's). The availability of increased yields of the mRNA's would, in turn, enhance production of the enzymes, or modifications thereof, by recombinant DNA production methods.
For a recent review of plasminogen activators, particularly t-PA, UK and SCU-PA, see Cederholm-Williams, S.A., "Molecular Biology of Plasminogen activators and recombinant DNA progress", Bio Essays, 1, 168-173 (1984).
Currently, the main (non-recombinant) source of t-PA in larger quantities is Bowes melanoma cells. These malignant cells yield sufficient t-PA for the purification and characterization of the enzyme and for preparation of monoclonal and polyclonal antibodies. Additionally, they produce sufficient specific messenger RNA (mRNA) to allow complete gene cloning. However, a source of t-PA and its messenger RNA from normal (i.e. non-malignant) cells is sought. Such a source is needed which will produce a large amount of t-PA from a serum-free medium and permit isolation of significant amounts of mRNA from the cells. The presence of serum greatly affects t-PA production and recovery due to the presence of t-PA inhibitor and a variety of proteins found in serum.
Two recent journal articles report on the production of PA from normal human cells. First, A. Kadouri and Z. Bohak, "Production of Plasminogen Activator In Cultures of Normal Fibroblasts", Biotechnology, June 1983, pp. 354-358, report on the production of plasminogen activator (PA) from several different strains of lung fibroblasts. They particularly studied the production of PA from the IMR-90 human diploid fibroblasts with different sera and with culture plates coated with poly-D-lysine. Two experiments were also reported in which a serum medium was used first, and thereafter, the medium was changed to a serum-free medium supplemented with 0.5 percent lactalbumin hydrolyzate for a few days production (see FIGS. 1(b) and 2(b) on page 356 thereof). A batch and a continuous production process were also studied, but the medium used for these experiments was not clearly identified. The article does not recognize that there are two distinct PA's produced by the cells.
In the second paper, Gerard C. Brouty-Boye et al., "Biosynthesis of Human Tissue-Type Plasminogen Activator By Normal Cells", Biotechnology, December, 1984, pp. 1058-1062, the authors report on the production of t-PA from human embryonic lung (HEL) cells in a serum-free medium. They studied the use of eight different possible inducers to stimulate the t-PA production from these cells. These potential inducers were calcitonin (salmon), cholera toxin, colchicin, concanavalin A, glycine, glycylglycine, heparin, lactalbumin, sodium butyrate, .alpha.-thrombin, and ultraviolet light. Of these, only concanavalin A significantly (4 times) enhanced production over that without an inducer. The authors also noted that no synergistic effects between concanavalin A and the other tested substances were seen.
Applicant's invention comprises the use of a combination of heparin and endothelial cell growth factor (ECGF) to enhance the production of t-PA and SCU-PA by normal human diploid lung fibroblast cells in a serum-free medium. ECGF is an extract from bovine hypothalmus or pituitary gland which stimulates the growth of bovine and human venous or aortic endothelial cells. (See Maciag, T. et al., Proceedings of the National Academy of Science, 76, pp. 5674-78 (1979) and Olander, J. et al., In Vitro, 16, p. 209 (1980)). ECGF is available commercially. Heparin at concentrations of 90 mcg/ml, has been reported to potentiate the stimulatory effect of ECGF on the proliferation of human umbilical vein endothelial (HUVE) cells and of endothelial cells from adult human blood vessels. No suggestion is made that these agents stimulate PA production by these cells. See Thornton, S. C. et al., Science, 222, p. 623 (1983). In this study the medium was supplemented with 20 percent fetal bovine serum. Maciag, T. et al., Science, 225, p. 932-5 (1984) have reported further that heparin has a strong binding affinity for ECGF as shown by the use of heparin-Sepharose chromatographic extraction of ECFG. Applicant's invention differs from the above in that the combination of ECGF and heparin has been found to significantly enhance the production of t-PA and SCU-PA by normal human lung fibroblast cultures in a serum-free medium. As described below, other endothelial cell polypeptide mitogens may be employed in this invention as endothelial cell growth factors, and, hence, hereinafter "ECGF" is used to denote endothelial cell growth factor itself, which can be isolated from bovine hypothalmus or pituitary gland. The words "an endothelial cell growth factor" as used hereinafter includes these other endothelial cell polypeptide mitogens.